Apparatus for separating by dielectrophoresis

ABSTRACT

The invention relates to a separator, which is particularly useful for separating cellular matter. The separator utilizes the phenomenon known as dielectrophoresis (DEP). 
     A DEP force effects a particle suspended in a medium. The particle experiences a force in an alternating electric field. The force is proportional to, amongst other things, the electrical properties of the supporting medium and the particle and the frequency of the electric field. 
     The separator, of the present invention, comprises a chamber (10) and a plurality of electrodes (12) disposed in the chamber (10). 
     An electric field established across electrodes subjects some of the particles to a stronger force than others such that they are confined within the chamber. Particles which are not confined are removed from the chamber by the supporting medium which is preferably urged through the chamber. Valves (101 and 202) are provided on exhausts of the chamber. 
     The invention is able to separate two different particles continuously.

FIELD OF THE INVENTION

This invention relates to improvements in separators and moreparticularly to improvements in dielectrophoretic separators.

BACKGROUND OF THE INVENTION

Dielectrophoretic (DEP) separators rely on the phenomenon thatsubstances within a non uniform DC or AC electric field experience adielectrophoretic (DEP) force. The (DEP) force causes the substance,which may gaseous, liquid, solid, or dissolved in solution, to movewithin the field.

A DEP field can have different effects upon different substances. Thiseffect has been used to filter or separate substances, usually solids insuspension, from a liquid for the purposes of analysis.

A study carried out by Gascoyne, Huang et al. and reported in "Meas.Sci. Technol. 3 (1992), at pages 439 to 445", describes the separationof mixed population of mammalian cells and more particularly theseparation of leukaemic cells from normal blood cells. However,separation was only achieved locally on electrodes.

A further study by Pethig, Huang et al. in J. Phys. D Appl. Phys. 24(1992) 881 to 888 describes an arrangement for positive and negativedielectrophoretic collection of colloidal particles usinginterdigitated, castellated microelectrodes. The arrangement describedenables a colloidal suspension to be separated locally. However,permanent separation of a colloid from the liquid in which it wassuspended was not possible.

U.S. Pat. No. 4,390,403 (Batchelder) describes and claims an arrangementfor filtering a species from a liquid. This describes a method whichemploys DC non-uniform electrical fields to manipulate one or morechemicals within a multi-electrode chamber so as to promote chemicalreactions between the chemical species.

German Offenlegungsschrift DE-A-4127405 purports to describe anarrangement for continuous separation of microscopic particles. It isstated that the arrangement overcomes the problem of convectional driftwithin a separator. The arrangement allegedly overcomes this problem byapplying a high frequency, electric travelling wave between rows ofelectrodes, which themselves are positioned between two additionalelectrodes which are electrically isolated from the aforementioned rowsof electrodes. The two additional electrodes (5 and 6 in FIG. 1) of thatdocument are arranged substantially parallel to one another. Thedescription of the aforementioned Offenlegungsschrift refers to "anadditional force field" which exists because of an electrophoreticeffect upon the particles. Electrophoresis relies upon particles beingcharged. The present invention utilises DEP only. Other examples offorces are mentioned. However, the disclosure is considered not to besufficiently clear and complete to be an enabling, in respect of these.

SUMMARY OF THE INVENTION

The present invention arose from a consideration of the problem ofpermanent separation of two substances, which may be in suspension in afluid, which may be a liquid.

According to a first aspect of the present invention there is providedapparatus for separating first and second particles from a fluidcomprising:

i) a first group and a second group of electrodes, which in use aredisposed in the path of the fluid supporting the first and secondparticles, such that the fluid may flow over the electrodes, theelectrodes being adapted to be placed in a filter chamber;

ii) the filter chamber having an inlet and at least one outlet;

iii) means for establishing a dielectrophoretic (DEP) field between thefirst and second groups of electrodes;

iv) the DEP field between the electrodes causing a resultant force to beexperienced by the particles, such that the first particles areconfined; and

v) means for selectively removing the second particles from the chamber.

Preferably control means is provided for establishing thedielectrophoretic field and for activating the means for selectivelyremoving the second particles from the chamber.

Preferably the control means comprises means for synchronizing one ormore valves located at the or each outlet of the filter chamber,arranged to permit fluid to exhaust from the chamber, with a respectivefluid pressurizing means.

Variation of the effect of the field is preferably achieved by varyingthe frequency of a signal applied across the electrodes. Differentfrequencies may be imposed simultaneously across different groups orsub-groups of electrodes.

A fluid pressurizing arrangement, which may be a pump, pressure sourcesyringe or even a gravity feed, may be used, in conjunction with theapparatus for causing or permitting the second particle to be urgedtowards a second outlet of the chamber.

The fluid pressurizing arrangement preferably comprises one or morepumps. Advantageously a pump is provided for each outlet of the chamber.Most preferably the or each valve is associated with one or more pumps,such that the synchronization means establishes a firstdielectrophoretic field for confining the first particles andsimultaneously opens a valve on an outlet of the chamber and causes thepressure of the interior of the chamber to exceed the pressure exteriorof the chamber. The result is that the second particles are exhaustedfrom the chamber. The control means then closes the valve and may allowthe pressure of the interior of the chamber to return to that pressureexterior of the chamber. Subsequently, or simultaneously, the controlmeans then switches off the dielectrophoretic field which confines thefirst particle. The control means then activates a second valve andpressurizing means to urge first particles towards an outlet, which ispreferably a different outlet than the outlet through which the secondparticles are exhausted. The first particles are then exhausted from thechamber. The control means then repeats the sequence in a cyclic manner.The control means may open a valve to achieve pressurization within thechamber or it may activate a pump.

The invention differs over the arrangement described in DE-A-4127405 inthat a so called travelling wave is not generated. That is, there is nosequential or cyclic switching between adjacent electrodes or sets ofelectrodes. Separation is achieved by the combined effects ofconfinement by the DEP field followed by pumping of the supportingmedium.

The chamber may be oriented in such a way that the second particles areremoved from the chamber under the influence of gravity. The firstparticles may be removed from the chamber after all of the secondparticles have been removed. This may be via the same outlet. However,the first particles are preferably removed via a different outlet. Aseparate fluid pressurizing arrangement may be used to assist removal ofthe first particles.

The first and second groups of electrodes may be sub-divided intosub-groups, such that, for example there may be several pairs ofseparate electrodes. Selective switching of these sub-groups ofelectrodes includes cyclic switching of adjacent pairs of sub-groups ofelectrodes. These pairs may overlap so that a second member of a pair atone switching step becomes the first member of a different pair in asubsequent switching step.

Preferably the first particles are moved relative to the or eachelectrode by the fluid in which they are supported.

It is understood that the term "switching" includes: varying thepotential difference between adjacent electrodes and/or sub-groups ofelectrodes; and/or varying the current passing through the fluid, whichis usually a liquid, between adjacent electrodes or sub-groups ofelectrodes and/or varying the frequency of the voltage and/or current.

In particular it is preferred to vary the frequency of the voltage as ithas been discovered that variation of the frequency of the voltage givesrise to different dielectrophoretic forces upon different substances.That is to say, two different substances A and B supported in suspensionin a liquid behave quite differently and experience different magnitudesof dielectrophoretic force depending upon the frequency of applicationof the DEP field in which the particles are situated.

Furthermore by arranging, in series, at least one frequency generator,connected to adjacent sub-groups of electrodes, it is possible tocyclically switch electrodes in order to selectively attract and/orconfine one or both substances A or B in different regions at differenttime intervals. One or more pumps may be used in combination with thisarrangement. The result is that a "sweeping" effect is achieved wherebya first particle is urged towards a particular outlet of the chamberwhilst the second particle is/are held within a DEP field. Such anarrangement may be used to separate one particle or substance from amixture of two or more particles or substances.

Electrodes can have a longitudinal cross section which is regular andmay be triangular, sinusoidal, sawtooth or square in shape. Preferablyadjacent electrodes are interdigitated and are of a square, castellatedcross section. Electrodes may be easily envisaged as having a transverseperiphery which is in the form of a regular square wave, castellatedprofile. Selective switching and variation of the dielectrophoreticfield between opposite (adjacent) electrodes is such as to cause spatialpartitioning of substances around different regions of electrodes.Electrodes are preferably interdigitated.

Certain forms of live cellular matter experience a different DEP forceto that experienced by the same type of dead cellular matter. Similarly,normal and cancerous cells may experience different DEP forces in thesame DEP field. The magnitude of the DEP force depends upon physicalcharacteristics of cellular structures such as: concentration andmobility of the ionic components. It has also been observed thatdifferent forms of proteins and chromosomes experience different DEPforces and the invention may be used to separate these.

By way of example only and for purposes of clarity, for the remainder ofthe specification live cellular matter will be referred to as particle 8or type B and dead cellular matter will be referred to as particle A ortype A. It will be understood that types A and B are analogous to thefirst and second types if particle referred to above. The aforementionedspatial separation, in one particular embodiment of the invention,causes cellular matter of the particle type A to accumulate aboutportions of a surface of castellated interdigitated electrodes which aregenerally within "troughs"; that is between digits of the same electrodeand on top of electrodes, and for cellular matter of particle type B toaccumulate between "peaks" of opposite (adjacent) electrodes. Theseaccumulations have been compared with "triangles" or "diamonds" and"pearl chains" respectively. In one arrangement the cellular matter typeA making up the "triangles", or "diamonds" which accumulate around the"troughs" of electrodes, and on top of electrodes, has experienced agenerally weaker attraction towards that portion of the electrodesurface than the other cellular matter type B has been attracted to itsrespective portion of electrode. The reason for this is because of thespatial distribution of the magnitude of the dielectrophoretic forcesinduced on the two types of particles and whether the particles areexperiencing positive or negative dielectrophoresis. This is describedin more detail below and with reference to the section entitled"theory".

A useful analogy to help visualize the aforementioned spatialdistribution of DEP forces around an electrode, is to envisage a threedimensional graph showing diagrammatically an overall view of spatialdistribution of DEP forces across the surface of a single electrode. Thesurface of the electrode is projected in the x-y plane. The magnitude ofthe dielectrophoretic (DEP) field experienced at a point in that plane,is shown on the z-axis. Such a surface is useful in envisaging therelative potential energies which are possessed by particles A and B.The surface can be seen to define regions of "hills" and deep andshallow "valleys". This is described below with reference to some of theFigures.

If particles A and B are visualized as spheres of the same volume theirrelative attractive/repulsive forces to the respective portions ofelectrodes are proportional to the heights of the "hills" and depths ofthe "valleys" from the plane z=o. As any system will tend to try toexist in its lowest energy state it will be appreciated that type Bcells may experience greater DEP forces, i.e. they are held "tighter"within deeper DEP "valleys" than are type A cells. Some spheres willtend to accumulate easily and quickly within a deep sided "valley" andare less likely to be dislodged therefrom. For example by a solutionflowing over the electrode surface. Other spheres however, willaccumulate in a relatively shallow valley and may be dislodgedrelatively easily therefrom.

According to a second aspect of the present invention there is providedan electrode for use in the apparatus for separating the first andsecond particles from a fluid comprising: an electrical contact forconnection to an electrical energy source which is controlled to changeits polarity; and a surface, adapted for use in the filter chamber.

Preferably the electrode is at least coated or formed from anelectrically conductive substance such as gold or platinum. However,other suitably inert metals, such as noble metals or even inertnon-metals may be used.

According to a further aspect of the present invention there is provideda method for selectively separating first and second type particles froma fluid comprising the steps of:

i) passing the fluid containing the particles over surfaces of at leasttwo electrodes;

ii) arranging the electrodes in such a manner that a dielectrophoreticfield established between electrodes is capable of confining particlesof the first type to a greater extent than particles of the second type;and thereby

iii) causing or permitting particles of the second type to move relativeto particles of the first type such that particles of the second typeare separated from particles of the first type.

By providing one or more outlets to a filter chamber (within which theelectrodes are placed), it is possible to remove fluid via a firstoutlet, which fluid is depleted in the first type (A) particle. Fluidmay be removed from a second outlet which is depleted in the second type(B) particle.

Separation of the first and second type particles from the fluid isenhanced by switching dielectrophoretic fields between adjacentelectrodes and selective pumping such that movement of the first typeparticles occurs in one direction whilst movement of the second typeparticles occurs in a different direction. These directions arepreferably in the direction of the respective outlets and are inopposite senses. Removal of the or each type of particle is enhanced byemploying a pump, syringe or other pressurising apparatus and urging thesupporting fluid in one or both of the desired directions. The chambermay be oriented in such a way that particles are urged in the desireddirection by gravity.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of, and methods of performing, the invention, will now bedescribed, by way of example only, and with reference to the Figures inwhich:

FIG. 1 shows viable yeast cells, suspended in 280 mM mannitol ofconductivity 40 mS.m⁻¹ collecting at an electrode under positivedielectrophoresis for an applied voltage frequency of 10 MHz;

FIG. 2 shows viable yeast cells, suspended in the same mannitolsolution, being repelled from the electrode under negativedielectrophoresis for an applied voltage frequency of 10 kHz;

FIG. 3 shows the time-averaged potential energy profile for a 3 μmradius particle suspended in an aqueous medium and experiencing positivedielectrophoresis;

FIG. 4 shows the potential energy profile for the same particle in whichit experiences negative dielectrophoresis;

FIG. 5 shows an overall view of an electrode divided, for calculationsof the surface charge density β, into 675 sub-areas contained within 12elements;

FIG. 6 shows an overall view of an interdigitated electrode;

FIG. 7 shows a time-averaged potential energy profile for a 3 μm radiusparticle, suspended in aqueous medium located in a plane 3.5 μm abovethe electrode surface;

FIG. 8 shows the potential energy profile for the same particle andelectrodes for the case of negative dielectrophoresis;

FIGS. 9 and 10 show the potential energy profiles of FIGS. 7 and 8respectively modified by superimposition of an extra translational forceof the order 1.5 pN;

FIG. 11 is a simplified diagrammatical view of part of a separatorarrangement;

FIG. 12 is an overall schematic view of the separator of FIG. 11 andshows frequency generators under the control of a computer;

FIGS. 13a to 13d illustrate diagrammatically, and in a simplifiedmanner, plan views of interdigitated electrodes which are part of theseparator of FIG. 11 and how these are used to separate two types ofparticles A and B;

FIG. 13a shows the beginning of a separation cycle, the DEP field isenergised;

FIG. 13b shows particles of type A being moved to the left by fluid flowwhile the DEP field strongly holds particles of type B;

FIG. 13c shows the DEP field switched off and all particles are moved tothe right by fluid flow;

FIG. 13d shows the dielectrophoretic field is re-established, particlesof type A are moved to the left, while particles of type B are stronglyheld;

Figure 14a shows an enlarged plan view of a portion of an interdigitatedelectrode;

FIG. 14b shows an enlarged plan view of portions of an interdigitatedelectrode pair and shows grouping of first and second cell types (A andB) around different portions of the electrodes;

FIG. 15a shows a graph of a three dimensional surface representingpositive dielectrophoretic field potential between adjacent electrodes;

FIG. 15b shows a graph of a three dimensional surface representingpositive dielectrophoretic field potential;

FIG. 16a shows a graph of a three dimensional surface representingnegative dielectrophoretic field potential;

FIG. 16b shows a graph of a three dimensional surface representingnegative dielectrophoretic field potential;

FIG. 17 is a view of a polynominal electrode, showing collection ofviable cells along electrode edges under positive dielectrophoresis andnon-viable cells in the center under negative dielectrophoresis;

FIG. 18a shows a graph depicting a three dimensional surfacerepresenting a positive dielectrophoretic field potential betweenadjacent electrodes and corresponding to the arrangement in FIG. 17;

FIG. 18b shows a graph depicting a three dimensional surfacerepresenting negative dielectrophoretic field potential betweenelectrodes in the arrangement of FIG. 17;

FIG. 19 shows a plan view of viable (living) and non-viable (dead)(methylene blue stained) yeast cells collected at electrodes afterapplying a 5V (pk--pk) 10 kHz signal;

FIG. 20 shows dielectrophoretic separation of viable and non-viableyeast cells using interdigitated, castellated electrodes and a 5V(pk--pk) 10 MHz signal;

FIG. 21 shows the viable cells which remain in the chamber afterflushing out the non-viable cells with the 10 MHz signal applied to theelectrodes;

FIG. 22 shows the dielectrophoretic spectra of viable and non-viableyeast suspensions as measured with a split-beam dielectrophoreticspectrometer;

FIG. 23 shows a schematic outline of an experimental system;

FIG. 24 shows a graph of percentage viability of mixed cell suspensionsdetermined by methylene blue staining and dielectrophoretic behaviour,versus the expected viability from the mixtures made;

FIG. 25 shows a graph of viability obtained from absorbance measurementsof an outflow of the filter chamber on selective flushing of first theviable and then the non-viable yeast cells, versus the viabilityexpected from the mixtures made (r=0.980); and

FIG. 26 shows a schematic view of a filter chamber with valves at eachof two outlets.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A brief discussion of the theory will now be described with reference toFIGS. 1 to 10 inclusive.

Theory

The basic theory and practice of using dielectrophoresis for theselective immobilization of bioparticles at an electrode has beenavailable for more than 15 years, Pohl H. A. (1978) DielectrophoresisCambridge University Press (Cambridge). Positive dielectrophoresis isemployed, where the particles are attracted to regions of electric fieldmaxima at electrode surfaces, as shown in FIG. 1. Although isolatedfield maxima cannot occur away from electrodes, Jones T. B. and Bliss G.W. (1977) J. Appl. Phys. 48 1412-17, it is possible to levitateparticles in free space or liquids using electronic feedback to maintaina balance between gravitational and dielectrophoretic forces, Jones T.B. and Bliss G. W. (1977) J. Appl. Phys. 48 1412-17, Jones T. B. andKraybill J. P. (1986) J. Appl. Phys. 60 1247-52, Kaler K. V. I. S. andJones T. B. (1990) Biophys. J. 57 173-82, Kaler K. V. I. S, Xie J-P,Jones T. B. and Paul R. (1992) Biophys. J. 63 58-69.

Negative dielectrophoresis can be employed to confine particles instable positions away from electrode structures. In this case particlesare induced to move away from high field regions as shown in FIG. 2. Bysuitable choice of electrode geometry it is possible to define thelocations of the electric field minima towards which the particles aredirected and eventually confined, Huang Y. and Pethig R. (1991) Meas.Sci. Technol. 2 1142-46, Pethig R, Huang Y, Wang X-B and Burt J. P. H.(1992) J. Phys. D: Appl. Phys. 25 881-8, Gascoyne P. R. C, Huang Y,Pethig R, Vykoukal J. and Becker F. F. (1992) Meas. Sci. Technol. 3439-45. Thus, by using both polarities of dielectrophoretic forces, itis possible to manipulate and entrap microscopic particles to a degreethat depends on the potential energy profiles associated with bothelectric field maxima and minima.

Procedures are described for deriving the depths and profiles of thepotential energy "wells" or "valleys" into which particles may bedirected using positive and negative dielectrophoretic forces generatedby microelectrodes of polynomial and castellated geometry. The resultsobtained are verified using test bioparticles (yeast, bacteria and bloodcells) and demonstrations are presented of how such bioparticles may beselectively confined and released from the energy wells, according tocell type or viability.

Experimental Details Materials

Yeast cells of Saccharomyces cerevisiae (strain R XII, obtained from theInstitute of Biophysics, Free University of Berlin) were grown at 30° C.in a medium of pH 5 containing 5% sucrose (Sigma), 0.5% yeast extract(Oxoid) and 0.5% bacteriological peptone (Oxoid). The cells wereharvested at around 18 hours in their growth phase and washed threetimes in 280 mM mannitol. Suspensions were made in 280 mM mannitol towhich sufficient NaCl had been added to raise the conductivity to 40mS.m⁻¹, as determined at 50 kHz using platinum-black electrodes and aHEWLETT PACKARD (Trade Mark) 4192A impedance analyser. Heat-treated cellsuspensions were also prepared by heating at 75° C. for ten minutes andwashing them in the same way as the viable cells. On staining withmethylene blue, Stoicheva N. G, Davey C. L, Markx G. H. and Kell D. B.(1989) Biocatalysis 3 245-55, this heat treatment was found to result ina majority (over 95%) of the cells becoming non-viable. Suspensions withroughly equal amounts of viable and non-viable cells were made by mixingin 280 mM mannitol, and the conductivity of such suspensions wasadjusted to 1 mS.m⁻¹ with NaCl.

Sheep blood was collected, and stored at 4° C., in a sterile vacutainer(Becton Dickinson, Oxford) containing lithium heparin as ananticoagulant. Erythrocytes were obtained by centrifuging the blood at100 g for 5 minutes, and they were washed three times in 320 mM sucroseplus 3 mg.m1⁻¹ glucose solution. The cells were then suspended insimilar sucrose+glucose solution, whose conductivity had been adjustedto 10 mS.m⁻¹ using NaCl.

Micrococcus luteus (syn. M. lysodeikticus) bacteria, Fleming strain 2665obtained from the Bakh Institute of Biochemistry, Moscow, were grown innutrient broth (Oxoid) at 30° C. and harvested by centrifugation at 100g for 5 minutes. The cells were then washed three times and finallyresuspended as for the erythrocytes in 10 mS.m⁻¹ sucrose+glucosesolution.

Electrodes

Microelectrodes of polynomial geometry, Huang Y. and Pethig R. (1991)Meas. Sci. Technol. 2 1142-46, and interdigitated, castellated, PethigR, Huang Y, Hang X-B and Burt J. P. H. (1992) J. Phys. D: Appl. Phys. 25881-8, Price J. A. R, Burt J. P. H. and Pethig R. (1988) Biochim,Biophys. Acta 964 221-30, were produced using photolithographictechniques described elsewhere, Price J. A. R, Burt J. P. H. and PethigR. (1988) Biochim, Biophys. Acta 964 221-30. These electrode types areshown in FIGS. 3 and 4; and 5 respectively, and were used to demonstratethe selective trapping and release of viable and non-viable yeast cells,erythrocytes and bacteria using both positive and negativedielectrophoresis. Electrodes of pin-plate geometry were alsoconstructed, and these were used to determine unambiguously (see FIG. 1)the polarity of the dielectrophoretic effect exhibited by the cells as afunction of the electric field frequency and suspending mediumconductivity.

Potential Energy

As first described by Maxwell J. C. (1891) A Treatise on Electricity andMagnetism, 3rd ed. Vol.1, Ch.ix, Clarendon Press, Oxford, when anexternal electric field is applied to a system consisting of a particlesuspended in a dielectric medium, charges are induced to appear at theparticle-medium interface so as to lend to this polarized particle theproperties of an electric dipole. The corresponding potential energy ofthe system is given by:

    W=-m·E                                            Eqn. (1)

where m is the induced effective dipole moment and E is the appliedfield. In this work we will restrict ourselves to interaction of theinduced dipole moment and the non-uniform external field, since theeffect of induced multipoles becomes dominant only in regions where thefield is zero, Washizu M. (1992) J. Electrostatics 29 177-88.

For a spherical particle of radius r, absolute complex permittivityε*_(p) (ε*_(p) =ε_(p) -jσ_(p) /ω, where σ is the conductivity and j=√-1) suspended in a medium of absolute complex permittivity ε*_(m) andsubjected to an A.C. electric field E(x,y,z)cosωt a_(z) of radianfrequency ω, the induced dipole moment is given, Huang Y, Holzel R,Pethig R. and Hang X-B (1992) Phys. Med. Biol. 37 1499-1517, by:##EQU1## where Re and Im refer to the real and imaginary components,respectively, of the Clausius-Mossotti factor f(ε*_(p),ε*_(m)) definedby: ##EQU2## Integrating equation (2) over times much longer than theperiod (2π/ω) of the applied field, then from equation (2) thetime-averaged potential energy of the polarized particle is:

    <W>=-2πε.sub.m r.sup.3 Re f(ε*.sub.p,ε*.sub.m)! E.sup.2 (rms)                                             Eqn. (4)

The dielectrophoretic force acting on the particle is given, Huang Y,Holzel R, Pethig R. and Wang X-B (1992) Phys. Med. Biol. 37 1499-1517,by

    F(ω)=2πε.sub.m r.sup.3 Re f(ε*.sub.p,ε*.sub.m)! ∇E.sup.2 (rms) Eqn. (5)

so that

    F(ω)=-∇ <W>                                 Eqn. (6)

which indicates that the dielectrophoretic force directs the particle toa region where its electrical potential energy is a minimum. Thus, for aparticle which is more polarizable than its suspending medium,corresponding to a positive value for the factor Re f(ε*_(p),ε*_(m))!,the particle will experience positive dielectrophoresis and be directedto a location where the local electric field (E²) is a maximum. Fromequation (2) this situation can also be understood to occur at thosefrequencies where the magnitude of the phase difference φ between theapplied field and the induced dipole moment is less than 90°.Conversely, a particle of sufficiently low polarizability to have anegative value for Re f(ε*_(p),ε*_(m))! (so that |φ| > 90°) will possessa minimum potential energy when directed to a local field minimum.

Thus, for selective dielectrophoretic manipulation and confinement ofparticles the important parameters to control are the electric fielddistribution (E, ∇E²) and the factor Re f(ε*_(p),ε*_(m))!. The fielddistribution is determined by the electrode geometry, whilst Ref(ε*_(p),ε*_(m))! varies with frequency according to the dielectricproperties (ε*_(p)) and (ε*_(m)) of the particle and surrounding medium,respectively. For mixtures of particles of differing dielectricproperties, selective manipulation can be achieved through suitablemodification of the conductivity or relative permittivity of thesuspending medium, whilst for particles of similar dielectric propertiesselectivity can be achieved using highly specific chemical treatments orattachments (eg antibody-antigen reactions) that change the dielectricproperties of one or more of the particle types.

Polynomial Electrodes

The basic polynomial electrode shape is shown in FIG. 3 and wasdesigned, Huang Y. and Pethig R. (1991) Meas. Sci. Technol. 2 1142-46,to provide a well defined spatial variation of the electric field. Thepolynomials defining the electric potential are derived from Laplace'sequations and are of the form

    f.sub.n (x,y)=af.sub.na +bf.sub.nb

where n defines the number of electrode pairs. Further details areprovided by Huang and Pethig as mentioned above and it is sufficient tostate that for the n=2 polynomial design of FIG. 3 the spatial variationof the field in the inter-electrode space is given by ##EQU3## where dis the radial distance between the centre of symmetry and an electrodetip, and V₁ and V₂ are the potentials applied to opposing electrodes.Thus, from equation (4), for an applied sinusoidal voltage V thetime-averaged potential energy of a particle suspended within thepolynomial geometry is given by ##EQU4## Three-dimensional plots of <W>are shown in FIGS. 3 and 4 for the specific case of d=64 μm. a particleradius r of 3 μm suspended in aqueous media where ε_(m) =80 ε_(O) andfor an applied voltage of 5 V (rms). The potential energy profile shownin FIG. 3 corresponds to the case where the parameter Ref(ε*_(p),ε*_(m))! has a value of +0.2, so that the particle is trappedin a steep-sided energy well at the electrode edges under the influenceof positive dielectrophoresis. In FIG. 4 the parameter Ref(ε*_(p),ε*_(m))! has a value of -0.2, and now the particle is directedinto a potential energy well at the centre of the interelectrode space.Since the field is zero at the centre, Huang Y. and Pethig R. (1991)Meas. Scd. Technol. 2 1142-46, then <W> is also zero and can be taken asa reference point.

For a 3 μm radius particle initially suspended in aqueous medium at anelectrode edge (eg x=64 μm, y=0) and for which Re f(ε*_(p),ε*_(m))! hasa value of -0.2, then on application of a 5V (rms) voltage we find fromequation (8) that the particle is directed into a potential energy wellof relative depth 918 eV. In other words the particle has to overcome apotential energy barrier of at least 918 eV to escape the electrodesystem. From equation (6) the average dielectrophoretic force acting onsuch a particle, as it moves from the electrode edge to the center, canbe calculated to be 2.3 pN.

Interdigitated Electrodes

The geometric form of the interdigitated, castellated electrodes isshown in FIG. 5. The dimensions (not to scale) of the electrodes areindicated. Charge interactions between the basic repeat structure andsix neighbouring ones on either side of the same electrode and theadjacent one of opposite potential were taken into account. Details ofthe electrode are described by Pethig R, Huang Y, Wang X-B and Burt J.P. H. (1992) J. Phys. D: Appl. Phys. 25 881-8. To derive the potentialenergy profiles for such electrodes, numerical computations of theelectric field distribution were made following the charge densitymethod Martinez G. and Sancho M. (1983) Am. J. Phys. 51 170-4, BirtlesA. B, Mayo B. J. and Bennett A. W. (1973) Proc. IEE 120, pp. 213-220,using a VAX (Trade Mark) computer and Fortran (VAX/VMS operationsystem).

The charge density method employs the following relationship between thepotential V(r) and charge density distribution p(r') on the electrodesurface S: ##EQU5## where ε_(m) is the absolute permittivity of thesurrounding medium, and r and r' are any points over S, which caninclude more than one electrode. The solution of equation (9) to findthe charge density function ρ(r') is facilitated by division of theelectrodes into sub-areas of such sufficiently small size that theirsurface charge densities can be assumed uniform. Dividing S into nsub-areas s_(j) (j=1,2, . . . n) of surface charge density ρ_(j),eluation (9) then takes on the matrix form; ##EQU6## Here, r₁ is thegeometrical centre of the sub-area s_(l) and X_(ij) is given by ##EQU7##From knowledge of the distribution of the sub-areas one can determineX_(ij), the potential at point r_(i) due to unity charge density onsub-area s_(j). The charge density ρ_(j) over the whole electrodesurface can then be calculated from the relationship

    ρ=X.sup.-1 V                                           Eqn. (12)

where ρ= ρ₁ ρ₂ . . . ρ_(n) ^(1'), X_(ij), =1 (X_(ij), =1, . . . n; j=1 .. . n), and V= V₁ V₂ . . . V_(n) 1'is the known potential applied to theelectrode. Having obtained the charge density ρ_(j) (j=1, . . . n) thepotential at any point r_(k) is found by substituting X_(ij) in equation(10) to give ##EQU8##

The interdigitated electrode design consists of a periodic"castellation" structure shown in FIGS. 5 and 6. For the calculations ofthe surface charge density the basic repeat structure was divided into675 rectangular sub-areas contained within elements 1-12 shown in FIG.5. Although the charge distribution within the 12 elements of the basicrepeat castellation structure might differ from each other the chargedensity on similar elements (ie identified by the same number) wereassumed to be the same. The relative sizes of the elements and thenumber of sub-areas within them, were chosen on the basis of preliminarycalculations of the surface charge distribution. Those regions (e.g.elements 7 and 10) of greatest charge density variation were allocatedthe largest number of sub-areas. Based on the assigned sub-division ofthe electrode surfaces, the potential coefficients (X_(ij)) werecalculated using equation (11) and the procedure described by Reitan andHiggins 17!. In the process of calculating matrix X, collectivecharge-charge interactions between sub-areas, located at different partsof the same electrode, as well as with those of an adjacent electrode,were taken into account. For example, referring to FIG. 5, the electricpotential at all sub-areas s_(ij) in element 7 were calculated takinginto account not only the charge densities occurring in the 675sub-areas of the basic castellation unit, but also those occurring inelements 1-12 for the next 6 castellations on the left and right handsides, as well as for those located on the adjacent electrode. Thecharge density distribution (675 values for the charge density at the675 sub-areas) was then obtained using equation (12) for assumedelectrode potentials of +1V and -1V applied to the (interdigitated)electrode pairs. An example of distance d1 shown in FIG. 5 is 320 μm.

From the derived charge density distribution, the electric potentialdistribution was obtained using equation (13). The field E (=-grad V)and the dielectrophoretic force factor ∇E² were then derived for pointsuniformly distributed on a plane located 3.5 μm above the electrodes,and the resulting three-dimensional plots of the time-averaged potentialenergy <W>, derived using equation (4), are shown in FIG. 7 and 8.

FIG. 7 depicts the situation for positive dielectrophoresis (Ref(ε*_(p),ε*_(m))!=+0.2), whilst in FIG. 8 the potential energy profilefor negative dielectrophoresis is shown (Re f(ε*_(p),ε*_(m))!=-0.2). Theother parameters used to derive these profiles are specified below. Therelative change in the absorbance of the yeast suspension is measuredafter the application of A.C. voltages to the electrodes. From theFigures it can be seen that under a positive dielectrophoretic forceparticles are directed into potential energy traps at electrode edges,irrespective of their initial locations within the electrode structure.However, with negative dielectrophoresis, particles initially located inthe inter-electrode space, are directed into energy wells in "bay"regions of electrodes, whilst those initially located above theelectrode surfaces are directed onto the surfaces of "tips" ofelectrodes.

From the results of FIG. 7 it is also evident that by comparison withthose confined in negative dielectrophoretic energy wells, particlestrapped under positive dielectrophoresis must overcome large potentialenergy barriers in order to escape the electrode system. The particlesexperience a positive dielectrophoretic force (Ref(ε*_(p),ε*_(m))!=+0.2) generated by interdigitated electrodes ofcharacteristic dimension 80 μm. The applied voltage is 5V rms, and theX-Y coordinates are specified in terms of the shown electrode geometry.This can be appreciated more clearly with reference to FIGS. 8 and 9,which show how the potential energy profiles are modified onsuperimposing an extra force field (e.g gravity or fluid flow) onto thedielectrophoretic forces. Particles under the influence of a positivedielectrophoretic force will be retained within deep energy wells,whereas for those experiencing negative dielectrophoresis the barriersrestricting their translational freedom over the electrode system arenot very large.

Results and Discussion

From equations (3) and (4) it follows that, for a suspension consistingof two particle types, with careful choice of the suspending mediumconductivity, it is possible at some frequency to attain the situationwhere the parameter Re f(ε*_(p),ε*_(m))! for each particle type is ofopposite polarity. This suggests a useful application, namely thecapability of separating the components of an heterogeneous suspensionusing dielectrophoretic forces. The following experiments were made toillustrate the feasibility of this.

Separation of viable and non-viable yeast cells using polynomialelectrodes

A 50 μl sample of a suspension of mixed viable and non-viable(heat-treated) yeast cells was pipetted onto a polynomial electrodestructure of dimension 128 μm between opposite electrode tips. 10seconds after applying a 10 MHz, 5 V (rms) signal to the electrodes thecollection pattern shown in FIG. 17 was observed. From methylene bluestaining tests and separate dielectrophoretic measurements on viable andnon-viable cells using the pin electrode system of FIG. 1, it wasconcluded that the result shown in FIG. 17 depicts viable cells beingcollected at the electrode edges and non-viable ones being confined tothe central inter-electrode region.

Thus, at a frequency of 10 MHz and in a suspending medium ofconductivity 1 mS.m⁻¹, viable and non-viable yeast cells exhibit apositive and negative value, respectively, for the factor Ref(ε*_(p),ε*_(m))!. This, in turn reflects differences in the dielectricproperties of the cell wall, membrane and cell interior of a viable andnon-viable yeast cell, as quantitatively described elsewhere (Huang Y,Holzel R, Pethig R. and Wang X-B (1992) Phys. Med. Biol. 37 1499-1517).Cells exhibiting a positive Re f(ε*_(p),ε*_(m))! value are directed tothe regions of greatest field intensity, whilst those of negative Ref(ε*_(p),ε*_(m))! become confined to the region of minimum E² value.

Separation of Erythrocytes and Micrococcus luteus using Interdiaitatedelectrodes

Samples of the erythrocyte and M. luteus suspensions were mixed togetherand a 50 μl sample of this mixture was pipetted onto an interdigitatedelectrode array of characteristic dimension 80 μm. A 5 V (rms), 10 kHz,signal was applied to the microelectrodes. The resulting distributionsof red blood cells and bacteria are similar to those shown in FIGS. 14aand 14b. As can be seen from these Figures, the blood cells (6 μmdiameter) collected as triangular aggregations in the electrode bayregions and in diamond-shaped patterns on the surfaces of theelectrodes, whilst the smaller bacteria collected at the electrodeedges. A small proportion (less than 5%) of the erythrocytes weretrapped by steric hindrance within the bacterial populations.

Measurements on the separate erythrocyte and bacteria suspensions, usingthe pin electrode system of FIG. 1, revealed that at 10 kHz and in the10 mS.m⁻¹ sucrose+glucose medium, the micrococci and erythrocytesexperienced positive and negative dielectrophoretic forces,respectively. This is in agreement with the earlier designations of thetriangular, diamond-shaped and pearl-chain collection patterns obtainedfor yeast cells when using the interdigitated electrodes (Pethig R,Huang Y, Wang X-B and Burt J. P. H. (1992) J. Phys. D: Appl. Phys. 25881-8).

The different behaviour of the blood cells and bacteria is primarilyrelated to the fact that the blood cells are bounded by lipid membranes,whilst the bacteria are bounded by heteropolysaccharide cell walls. At afrequency of 10 kHz the blood cell membranes appear more resistive thanthe 10 mS.m⁻¹ suspending medium (ie Re f(ε*_(p),ε*_(m))! is negative)and so they experience a negative dielectrophoretic force. The cellwalls of the bacteria, on the other hand, have electrical propertiessimilar to ion exchange resins and are relatively conducting (ieRe(f(ε*_(p),ε*_(m))! is positive). The micrococci therefore experiencethe potential energy profile of FIG. 7, whilst FIG. 8 corresponds to thesituation for erythrocytes experiencing negative dielectrophoresis (Ref(ε*_(p),ε*_(m))!=-0.2).

The collection patterns obtained are thus in good agreement with thoseexpected when the blood cells and bacteria rearrange themselves so as tominimize their potential energies.

Finally, the result shown in FIGS. 9 and 10 indicate that particlesretained by a negative dielectrophoretic force are more easily releasedthan those held by positive dielectrophoretic forces. This was verifiedby flushing liquid over the electrode array. After separation of themicrococci and red blood cells using a 5V rms (10 kHz) signal, and withthis signal maintained, the blood cells were removed by the flowingliquid, whereas the bacteria remained firmly trapped at the electrodeedges. On removing the voltage signal, the bacteria could then beflushed away. A similar result was obtained for a mixture of viable andnon-viable yeast cells in 1 mS.m⁻¹ mannitol solution. A 5 V (rms), 10MHz, signal resulted in the viable cells being trapped at the electrodeedges and remaining there under exposure to a cross-flow of liquid,whereas the non-viable cells, which initially collected in similardiamond-shaped and triangular-shaped aggregations for erythrocytes, wereswept away.

Conclusions

In previous work as mentioned above, it was demonstrated that electrodesof polynomial and interdigitated, castellated geometry can facilitateparticle collection arising from both positive and negativedielectrophoretic effects. A theoretical explanation was presented interms of the electric field patterns generated by the electrodes. Wehave extended this here to consideration of the potential energysurfaces experienced by particles subjected to dielectrophoretic forces.Furthermore, we have demonstrated that by careful choice of theconductivity of the suspending medium it is possible to find a frequencyrange where the different particle types in an heterogeneous suspensionare directed into spatially separated potential energy wells, accordingto the polarity of the dielectrophoretic forces acting upon them. Goodagreement between theory and experiment was obtained concerning thecollection patterns observed using the polynomial and interdigitatedelectrodes and the locations and geometric form of the potential energysurfaces.

For the case of the interdigitated, castellated electrodes it has beenfound that particles trapped in potential energy wells under the actionof negative dielectrophoresis can be more easily removed from theelectrode structure (e.g by fluid flow or gravitational forces) thanthose trapped under positive dielectrophoresis. Such selectiveconfinement and release of the different particle types in heterogeneoussuspensions can be envisaged to have interesting applications in thebiomedical and biotechnological sciences.

One way in which the invention may be performed will now be describedwith specific reference to FIGS. 11 to 18 inclusive.

Referring briefly to FIGS. 11 and 12, a filter or separator showngenerally at 10 comprises an array of electrodes 12 (shown in detail inFIG. 13) housed within a reservoir or chamber 14. The chamber 14 has aninlet 16 and a first outlet 18 and a second outlet 20. A pump 22 pumps asolution (not shown) into the chamber 14. The solution contains amixture of cells A and B. The mixture comprises living or viable cells Band dead or non-viable cells A. These cells A and B are of the same cellvariety.

The solution passes over the array of electrodes 12 and the cells A andB are subjected to different dielectrophoretic forces depending onwhether they are alive or dead. The forces affect the resultant movementof cells A and B within the chamber 14. The resultant effect is that Atype cells are urged towards the first outlet 18 and that B type cellsare urged towards the second outlet 20. However, several steps areinvolved in the separation process and these are described in detailwith reference to FIGS. 13a to 13d below.

Pumps 23 and 24 are used to pump the liquid supporting the cellsbackwards and forwards within the chamber 14. The pumps 23 and 24 mayalso pump liquid rich in A type or B type cells respectively to furtherfiltering chambers (not shown) in order to concentrate the cellsfurther. It will be appreciated that a cascade of filters or separatorsmay be connected together in series to enable the separation of morethan two different species of cell, protein or any other substance whichexperiences a DEP force within a DEP field. In addition separate inlets26 and 28 may be optionally provided to allow a different, inert mediumto pass through the filtering chamber 14 and collect the A and B typecells. However, it is appreciated that this is not required butoptional. The liquid supporting the two types of cell enters via inlet16 under pressure of pump 22.

Four frequency generators 30, 32, 34 and 36 are linked to selectedsub-groups of electrodes 30A, 32A, 34A and 36A respectively within thechamber 14 and are controlled by computer 38. It will be appreciatedthat a single frequency generator may be used instead of four separatefrequency generators. The single frequency generator may be connected toan amplifier (not shown). Pumps 22, 23 and 24 are also controlled by thecomputer 38. The frequency generators 30, 32, 34 and 36 are switched soas to vary the dielectrophoretic fields between the electrodes therebycausing different DEP forces to be applied to cell type A and cell typeB. The cells A are confined to triangular regions whilst the cells B areattracted by strong DEP forces to the electrode surfaces. Pumps 23 and24 are then used, alternatively, to urge fluid in one direction or theopposite direction as described below. The overall result is that liquidexhausting from the second outlet 20 is richer in cell type B than thatexhausting from the first outlet 18; and liquid exhausting from thefirst outlet 18 is richer in cell type A than that liquid exhaustingfrom the second outlet 20. This is explained generally with reference toFIGS. 13 to 18 below.

FIGS. 13a to 13d show views of a portion of an electrode array 12 infour sequential instances of time, although the time intervals may notnecessarily be equal. A mixture of cell types A and B is introduced intothe chamber 14. A dielectrophoretic field is applied which-attracts celltype B to a greater extent than cell type A to particular portions ofthe electrode. FIG. 13a shows an initial instant at which cells of typeA and type B form separate patterns between adjacent electrodes 42 and43. The views in FIGS. 13a to 13d show three pairs of electrodes 40 and41; 42 and 43; and 44 and 45. A dielectrophoretic field tends toseparate the cell types A and B such that cell type B forms chains,which are herein referred to as pearl chains, between "peaks" or "tips"of oppositely facing electrodes 42 and 43. Cell type A tends to formaround surfaces of electrodes 42 and 43, and within "troughs" or "bays"of oppositely facing electrodes, into triangular or diamond patterns.The grouping of the two different types of cells is explained above inthe section headed "Theory" although brief reference is made to thephenomenon, from an energy point of view, below with reference to FIGS.15 to 18.

FIG. 13b shows what occurs whilst the dielectrophoretic field ismaintained between the electrodes 42 and 43 and when liquid supportingthe cells A and B is urged through the chamber 14 by pump 23. The A typecells are forced (to the left) in the direction of outlet 18 as they areheld by weaker DEP forces. The B type cells remain attached to thesurfaces of electrodes as they are held by relatively stronger DEPforces. Thus cell type A moves in a direction of electrode 41 whilstmaintaining cell type B, in situ between electrodes in "pearl chains".

FIG. 13c shows a subsequent instant when the dielectrophoretic field isswitched off. Liquid via inlet 16 is introduced under pressure by pump22. Both cell types A and B are moved to the right in the direction ofoutlet 20. The DEP field is then re-established.

FIG. 13d shows the DEP field switched on. It is appreciated that the Atype and B type cells have been displaced (by one electrode pair)towards exit 20 (i.e. towards the right hand side of the page). B typecells are now attracted to electrodes 43 and 44 in the DEP field. Theseare different electrodes from those to which the B type cells werepreviously attracted. In general the electrodes will be to the right ofthe electrodes.

Pump 23 then urges fluid towards exit 18 and in doing so type A cellsare also moved towards exit 18. The overall result is that the two celltypes A and B are spatially divided. At each further step of spatialdivision the concentrations of cell types A and B become purer thecloser they are to respective outlets. Cell type B, trapped withinclusters of cell type A, become randomly dislodged and are urged towardsthe relevant outlet, and vice versa. This also has the effect ofimproving separation.

A fresh charge of solution containing cell types A and B is thenintroduced into the separator between electrodes 42 and 43 and theprocess is then repeated such that subsequent cycles of switching giverise to continuous resultant displacement of cell type A towards exit 18and cell type B towards exit 20. The concentration of each cell typebecomes purer at each step.

FIG. 14a is an enlarged view of cells accumulating around a surface ofan electrode 42, the triangle of A type cells being shown in the"troughs" of electrode 42.

FIG. 14b is an enlarged view between two electrodes 42 and 43 and showsthe "pearl chains" of cell types B between "peaks" of electrodes and thetriangular shapes of cell type A.

FIGS. 15a, 15b, 16a and 16b show diagrammatically the steep sided deeppotential energy "wells" or "valleys" in which cell types B arecollected. The analogy of the depth of "wells" or "valleys" is thatdescribed above. Cell type B "falls" into a relatively deep "valley",whereas cell type A tends to accumulate at the summit of hills fromwhere they are easily removed.

One particular experiment is described in detail below and withreference to FIGS. 19 to 25 and illustrates the effectiveness of thefilter or separator in separating live and dead cells of a particularcell variety. An experimental station, as depicted in FIG. 23, was usedas a batch separator to separate two types of cells. Efficiency ofseparation was then measured by absorbance techniques, methylene bluestaining and plate counts.

Brief Summary of Experiment

Dielectrophoresis, the movement of particles in non-uniform electricfields, was used to rapidly separate viable and non-viable yeast cellswith good efficiency. Known mixtures of viable and heat-treated cells ofSaccharomvces cerevisiae were separated and selectively isolated usingpositive and negative dielectrophoretic forces generated bymicroelectrodes in a small chamber. Good correlations with the initialknown relative compositions were obtained by direct microscopic countingof cells at the electrodes after initial dielectrophoretic separation(r=0.995), from methylene blue staining (r=0.992) and by opticalabsorption measurements (r=0.980) of the effluent after selectivelyflushing out the viable and non-viable cells from the chamber. Throughmeasurement of cell viability by staining with methylene blue and platecounts, for an initial suspension of ca. 1.4×107 cells m1⁻¹ containing60% non-viable cells, the dielectrophoretically separated non-viablefraction contained 3% viable cells and the viable fraction 8% deadcells. The separation efficiency is increased by dilution of the initialsuspension or by repeat operation(s). Cell viability was not affected bythe separation procedure.

The determination of cell viability is not straightforward and resultsare often very dependent on the technique employed. However, suchdetermination is of considerable practical and theoretical importance(Jones, 1987; Higgins, 1992; Kaprelyants and Kell, 1992) and thedevelopment of new techniques for the study of cell death, as well asfor the physical separation of viable and non-viable cells in a mixedpopulation, would be very useful. The phenomenon of dielectrophoresis iscapable of providing the basis for such techniques.

Dielectrophoresis (DEP) is the movement of particles in non-uniform ACelectric fields, the theory and practice of which is well documented(Pohl, 1978a & b; Pethig, 1979, 1991). As a result of an externallyimposed electric field a dipole moment is induced in the particle(cell), and if the field is non-uniform the particle experiences a nettranslational force which may direct it either towards or away from highfield regions. This induced motion constitutes the DEP effect, and forcells is comprised of several frequency-dependent components (Burt etal., 1990; Pethig 1991; Pethig et al., 1992).

Below around 1 kHz the effect is largely controlled by polarizationsassociated with surface charge effects, whilst between 1 kHz and 1 MHzsurface conduction, dipolar relaxations at membrane or cell wallsurfaces, membrane fluidity, as well as transmembrane ion transportprocesses, are dominant influences. Above 1 MHz the controllinginfluences on the DEP response are membrane capacitance and interfacialpolarizations associated with surface and internal cell structure. Themain variables under the experimenter's control are the conductivity andpermittivity of the suspending medium and the frequency of the appliedfield. Thus, it is possible to choose the variables such that a mixtureof particles with different DEP properties can be separated, and this isgreatly facilitated using microelectrodes of an interdigitated,castellated, design (Price et al., 1988; Burt et al., 1989, 1990; Pethiget al., 1992).

It has already been shown (Pohl, 1978a & b; Huang et al., 1992) that theDEP properties of viable and non-viable yeast cells are significantlydifferent, and differences have also been reported using the closelyrelated techniques of dielectric spectroscopy (Boulton et al., 1989;Stoicheva et al., 1989; Markx et al., 1991) and electro-rotatlion(Holzel and Lamprecht, 1992; Huang et al., 1992). The DEP method used byPohl (Pohl and Hawk, 1966; Crane and Pohl, 1968; Pohl, 1978a & b) andMason and Townsley (1971) to separate cells employed one-electrode (andcounter-electrode) only and did not provide a good efficiency inseparation. The method described below employs two new features toachieve a high efficiency of separation. These are the use ofinterdigitated microelectrode arrays and the controlled application ofboth positive and negative dielectrophoretic forces. Also, the method isin principle generic since the dielectrophoretic properties can varyconsiderably between cells of different organisms, and indeed is alsodependent on physiological states other than the viability (Mason andTownsley, 1971; Pohl, 1978a & b; Pethig, 1991; Gascoyne et al., 1992).

The dielectrophoretic separation method described here operates on thebasis, as described above (Huang et al., 1992). That is frequency rangescan be found where: (i) Both viable and non-viable yeast cells exhibitpositive DEP and (ii) Viable cells exhibit positive DEP and non-viablecells negative DEP. The other phenomenon exploited is associated withthe fact that when using interdigitated, castellated microelectrodes,cells collected under positive DEP are held in deep and steep-sidedpotential energy wells at electrode edges; whereas under the influenceof negative dielectrophoretic forces, the cells are retained astriangular-shaped aggregations in shallow potential energy wells(Gascoyne et al., 1992; Pethig et al., 1992). Thus, cells attracted tothe electrodes by positive DEP are not easily dislodged by flushingfluid over the electrodes, whereas those cells retained by negative DEPare readily and selectively removed by such action.

METHODS Yeast:

The yeast used was baker's yeast (Saccharomyces cerevisiae, strain RXII,obtained from the Institute of Biophysics, Free University of Berlin)grown at 30° C. in a medium of pH 5 consisting of 5 g 1⁻¹ yeast extract(Oxoid), 5 g 1⁻¹ bacterial peptone (Oxoid) and 50 g 1⁻¹ sucrose. Theyeast was grown overnight, harvested and washed 4 times in 280 mMmannitol. The cells were rendered non-viable by heating to 90° C. in awaterbath for twenty minutes, after which they were washed as before.Suspensions with different relative amounts of viable and non-viablecells were made by mixing.

Dielectrophoretic spectrometer:

The DEP spectra of suspensions of viable and of non-viable yeast cellswere measured so as to ascertain the frequency ranges where the viableand non-viable cells exhibited either positive or negative DEP.Suspensions of viable and non-viable (heat treated) yeast cells wereprepared having an absorption of 0.6 at 655 nm in a cuvette of 1 cm pathlength (corresponding to 1.4×10⁷ cells m1⁻¹), and their DEP spectra wereobtained using a split-beam spectrometric system, based on a previousdesign (Price et al., 1988; Burt et al., 1989, 1990). One component ofthe split laser beam monitored the optical density of the cellsuspension located between two interdigitated electrode arrays, of thesame geometry as those used in the cell separation chamber. The othercomponent of the split-beam corrected for random fluctuations of thebeam intensity and also provided a reference signal to give increasedsensitivity of measurement. Positive DEP manifested itself as areduction in optical density of the cell suspension, whilst the effectof negative DEP was to increase the optical density as a result of cellsbeing repelled away from the electrodes into the bulk suspendingsolution. As described elsewhere (Price et al., 1988; Burt et al., 1989)the initial rate of change of the optical absorbance, on application ofthe AC voltage signal to the electrodes, is proportional to the DEPcollection rate of the cells.

Dielectrophoretic separation:

The cell separation chamber incorporated interdigitated, castellatedmicroelectrodes of the same basic design and construction as those usedin DEP studies of colloidal particles, bacteria, yeast and mammaliancells (Burt et al., 1989, 1990; Price et al., 1988; Pethig et al.,1992). The electrodes were fabricated onto a microscope slide and thecharacteristic dimension defining the castellated geometry was 80 μm.The chamber, of volume 50 μl, was constructed by placing a polyacetatespacer and a microscope cover slip on top of the electrodes, and sealingthe system with epoxy resin. The cells and suspending fluid are injectedinto and flushed from the chamber through two small diameter tubes. Thefirst stage of the separation process consisted of applying to theelectrodes a sinusoidal voltage of such a frequency that both the viableand non-viable cells collected at the electrode tips as a result of apositive dielectrophoretic force. With this voltage signal stillapplied, the chamber was then flushed through with clean suspendingfluid so as to remove cellular debris and cells not captured by theelectrodes. The frequency of the applied voltage was then adjusted sothat the non-viable cells redistributed themselves so as to collect intriangular aggregations at the electrode bay regions under the influenceof a negative dielectrophoretic force, whilst the viable cells remainedat the electrode tips under a positive force. With this voltage signalstill applied, the chamber was then flushed through to selectivelyremove the non-viable cells from the chamber. The final stage involvedswitching off the applied voltage to the electrodes and flushing thechamber in order to remove the viable cells.

Measurement of the separation of cells of different viability wasaccomplished in two ways. In the first method the cells were broughtinto the chamber by injection, a 5 Volt (pk--pk) 10 MHz voltage wasapplied to the electrodes and the number of cells occurring intriangular aggregations and on top of the electrodes, and of thosecollected at the electrode edges, were counted by direct microscopicobservation and from photographs of areas representative for theelectrode arrays. To compensate for the fact that some cells werepresent in the chamber from previous experiments, cell counts were alsomade before introducing the new sample.

In the second method cells were brought into the cell by injection andcollected at the electrode edges by applying a 10V (pk--pk) 10 kHzsignal. Non-captured cells and any cellular debris were flushed out with280 mM mannitol. The signal was then changed to 10 V (pk--pk) 10 MHzwhich had the effect of causing non-viable cells to migrate intotriangular aggregations and on top of the electrodes, whilst leaving theviable ones located at the electrode edges. By passing a gentle streamof fluid medium through the DEP chamber with the 10 MHz signal applied,the non-viable cells were selectively removed from the chamber. Thepassage of these cells was monitored as an increase of opticalabsorbance at 500 nm, using a 1 cm flow-through cell and a Pye-UnicamSP6-400 (Trade Mark) spectrophotometer. On removal of the non-viablecells, the voltage was switched off and the subsequent flushing of theviable cells from the electrode edges was also recorded as an increasein optical absorbance. The absorbance signal was followed in time andthe area under the two absorption peaks was measured. The flow ratethrough the chamber was 30 ml hr⁻¹, and suspensions of viable andnon-viable yeast cells of the same concentration exhibited the sameabsorbance at 500 nm.

Estimation of Viability:

To estimate the viability of cells, they were stained with methyleneblue (Stoicheva et al., 1989), and they were plated out on platescontaining growth medium with 1.2% agar.

RESULTS AND DISCUSSION

The DEP spectra of suspensions of viable and non-viable yeast cells,measured using the split-beam spectrometer, are shown in FIG. 22. Thesespectra provided the information required to enable the conditions forcell separation to be established, namely that both the viable andnon-viable cells exhibit a positive DEP of similar magnitude at 10 kHz,whilst above 2 MHz the non-viable cells exhibit a negative DEP effectand the viable ones a positive effect.

The result of applying a 5 V (pk--pk) 10 kHz voltage signal to theelectrodes for a suspension containing both viable and non-viable cellsis shown in FIG. 19. Both cell types collect (within 10 secs) at theelectrodes. FIG. 20 shows the result of changing the frequency of theapplied voltage to 10 MHz. The viable cells remain collected at theelectrode edges and in "pearl chains" between the "peaks" of electrodes,whilst the non-viable cells have rearranged themselves intotriangular-shaped aggregations in the electrode "bay" or "trough"regions. The non-viable cells are also collected onto the surface of theelectrodes away from the electrode edges and, although not fullyunderstood, this is considered to occur mainly under the influence of anegative dielectrophoretic effect (Pethig et al., 1992). Thisrearrangement of the cells is completed within 30-60 seconds. The twotypes of cell were thus easily recognisable and physically separated ona local scale by application of the 10 MHz signal. Observations usingmethylene blue treated cell suspensions confirmed that the stained cellscollected in the triangular formations and on top of the electrodes,whereas the unstained (hence viable) cells collected at the electrodeedges and in pearl chains.

The relative numbers of viable and non-viable cells were obtained bydirect microscopic inspection, as well as from photographic records, ofcell collection at the electrodes as seen in FIG. 20. FIG. 23 showsdiagrammatically how cells were syringed into the DEP separation chambercontaining the microelectrodes, and after DEP separation theirflushing-out was monitored by optical absorption. Cell viability wasdetermined using methylene blue staining. FIG. 24 shows the measuredcell viability versus the viability expected from the known compositionof the cell mixtures. Good correlations can be seen (correlationcoefficient r=0.992 and 0.995 for methylene blue staining anddielectrophoresis, respectively).

The cells were also separated by flushing the DEP chamber as describedabove, so as to first selectively remove the non-viable cells (FIG. 21)and then the viable cells. The relative numbers of negative DEPcollected (non-viable) and positive DEP collected (viable) cells weredetermined by optical absorbance measurements. Previous studies (Burt etal., 1989) have shown for yeast concentrations up to around 1.4×107cells m1⁻¹ that the optical absorbance in 1 cm path length cuvettesvaries linearly with concentration (i.e. Beer's law is obeyed). Apartfrom the linear relationship between cell concentration (checked forviable and non-viable cell suspensions) the advantage of operatingwithin Beer's law is that errors associated with multiple lightscattering are avoided. In this work cell concentrations above 1.4×10⁷m1⁻¹ were not used. The results obtained are shown in FIG. 25, and areasonable correlation is seen (r=0.980) with the initial known relativecompositions of the suspensions. Examples of distances d2 through d5shown in FIG. 21 are 1 cm, 40 μm, 1 μm, and 1 cm, respectively.

After DEP separation of a suspension prepared using 40% viable and 60%non-viable (heat treated) yeast cells, the two separated components werestained with methylene blue and plated-out on growth medium with 1.2%agar. Viable cells (3%) were still present in the fraction supposed tocontain non-viable cells, whilst the fraction containing mainly viablecells also contained dead cells (8%). This shows that at the relativelyhigh cell concentrations used in these experiments (ca 10⁷ m1⁻¹) theseparation was not 100% successful. At these concentrations non-viable(stained) cells were sometimes trapped or sterically hindered by theviable cells at the electrode edges. This effect was reduced ifsuspensions of lower density cells were used. On plating out good growth(cell recovery 100% to within experimental error) was obtained fromfractions with viable cells, whilst only very few (3%) colonies wereobtained from fractions containing non-viable cells. It was found thatthe yeast viability was not affected by the applied electric field inaccord with earlier work of Forster and Emeis (1985) who demonstratedthat the viability of the even more fragile yeast protoplasts isunaffected by dielectrophoresis.

FIG. 25 shows a graph giving good correlation for both methods(correlation coefficient r=0.992 and 0.995 for methylene blue and DEP,respectively;

CONCLUSIONS

From analyses of the dielectrophoretic and electrorotational behaviourof yeast cells, Huang et al. (1992) showed that the cytoplasmic membraneconductivity of the cells increased on heat treatment from 2.5×10⁻⁷ Sm⁻¹ to 1.6×10⁻⁴ S m⁻¹, in parallel with a decrease of the internal cellconductivity from 0.2 S m⁻¹ to 7×10⁻³ S m⁻¹. These changes in cellularelectric properties give rise to the differences in dielectrophoreticbehaviour described here and form the basis of the separation technique.

The process of injecting cells into the separation chamber, trapping thecells using a 10 kHz signal and locally separating the viable fromnon-viable cells at the electrodes using a 10 MHz signal, can beachieved within 2 minutes. The measurements in which the numbers ofviable and non-viable cells were counted at this stage ofdielectrophoretic separation were made here by simple countingprocedures, but this can be automated using image analysis techniques(Gascoyne et al., 1992). This procedure can therefore provide a rapidmethod for ascertaining cell viability, without the need for chemicaltreatment of the cells, and for selectively collecting the cellsafterwards.

For 1.4×10⁷ cells m1⁻¹ of 40% viability, a significant number (8%) ofdead cells appeared in what should have been the fraction containing theselectively flushed-out viable cells alone. From direct microscopicobservations of the DEP effect on methylene blue treated suspensions,this "contamination" was found to occur because non-viable cells weresterically hindered and even trapped by the viable cells. This effectwas reduced significantly on 10-fold dilution of the initial suspension.Improved efficiency of separation can also be achieved by passing thecells through two or more stages of dielectrophoretic separation.Similar and/or other advantages may be gained from other microelectrodestructures and geometries.

Finally, preliminary data with stationary cultures (data not shown)indicate that cells at different physiological states can be identifiedthrough their dielectrophoretic behaviour, and the behaviour of moribundcells may be different from that of both viable and non-viable cells.Apart from the potential for selective cell separation technologies, acomparison of the dielectrophoretic technique with staining methods fordetermining cell viability and physiological state could thus provescientifically rewarding.

Another embodiment of the invention is described with reference to FIG.26.

As high field strengths are necessary to observe dielectrophoresis, theeffect is generally only observed on small scales using electrodes ofthe same order of size as the particles under investigation, at whichsuch field strengths can be easily generated. However, as a consequenceof the fact that the distance between the electrodes is very small theparticles usually only move over short distances, and unless one usesmany adjacent electrodes which are addressed consecutively (Burt &Pethig, 1990; Washizu et al., 1993), other forces such as thosegenerated by a flowing liquid or gravity are needed to move cells overlarger distances. Lin and Benguigui (1982) used interdigitatedelectrodes without castellations to separate inorganic particles fromthe flowing liquid. They did not attempt to separate particles withdiffering electrical properties nor did they attempt to make theirsystem continuous. Markx et al. (1993) showed the separation of viableand non-viable yeast cells using interdigitated castellated electrodes,but no attempt was made to achieve continuous separation. Althoughcontinuous dielectrophoretic separation has been attempted before usingconcentric cylinders (Mason & Townsley, 1971) or so-called isomotiveelectrodes (Pohl, 1978a & b) to generate the dielectrophoretic force,the results were rather unsatisfactory and yields were very low. Acyclic counterflow regime in a chamber containing arrays ofinterdigitated castellated electrodes 40, with which an efficientcontinuous separation can be achieved, is described below with referenceto FIG. 26 and FIGS. 13a to 13d. As a model system viable and non-viableyeast cells were taken.

Materials and Methods Cells

The yeast cells used were, Saccharomyces cerevisiae strains RXII,obtained from the Free University in Berlin. The yeast was grown asdescribed before (Markx et al., 1990), harvested and washed 4 times indeionised water. Non-viable yeast cells were obtained by heat treatment(20 min a 90° C.), and washes as described before. Non-viable and viablecells were then mixed in the ratio 50%--50% . The viability of the yeastcells was tested using methylene blue staining (Stoicheva et al., 1989).The optical density of the suspension used was 0.288, corresponding to acell concentration of 7 E6 cells m1⁻¹.

Apparatus

The dielectrophoretic separation chamber is shown in FIG. 26. Theinterdigitated, castellated electrodes (made from gold on a chrome base,with a length of 20 mm, characteristic dimension of the castellations 70μm) were fabricated on top of 12, 26 mm wide and 76 mm long microscopeslides using photolithographic techniques. The microscope slides wereglued on top of a glass plate. Connections to the electrodes on themicroscope slides were made by soldering. A chamber was constructedabove the electrodes using a 200 micron PTFE spacer and furthermicroscope slides. Liquid was pumped in and out of the chambers through1 mm inner bore PVC and silicone tubing. Cells were pumped in throughthe tube in the centre of the chamber, whilst fresh liquid without cellswas pumped in through tubes at the two ends of the chamber, and liquidcontaining separated cells pumped out through two different tubes at thefar ends of the chamber. The whole system was sealed using flowablesilicone rubber (RS) and is sterilisable.

An outline of the complete steps of separation is shown in FIGS. 13a to13d. 13a. The cells are brought in and the voltage is applied. Viablecells are attracted to high field regions between the electrodes, whilstnon-viable cells are repulsed. 13b. A gentle fluid flow dislodgesnon-viable cells and moves them in one direction. The viable cells arestill held. 13c. The applied voltage is set to zero. Both viable andnon-viable cells are moved in the opposite direction. 13d. The voltageis applied again and the non-viable cells are moved again in the samedirection as in b.

Peristaltic pumps (Gilson Minipuls 3 (Trade Mark)) and valves made fromsolenoids (RS) were used to control fluid flows. The flow rate of thepumps was in the order of 5.5 ml min⁻¹, AC voltages were applied by aFarnell LFM3 (Trade Mark) and a Krohn-Hite model 2000 (Trade Mark)frequency generators through a relay. The whole system wascomputer-controlled.

Continuous separation was achieved using the valve control regime shownin Table 1.

                  TABLE 1                                                         ______________________________________                                        Switching regime for continuous dielectrophoretic separation.                            Period 1                                                                            Period 2  Period 3                                                                              Period 4                                   ______________________________________                                        Applied voltage                                                                            0V      10V       0V    10V                                      Pump 100     on      off       off   off                                      Pump 200     off     on        off   on                                       Pump 300     off     off       on    off                                      Valve 101    on/off* on        off   on                                       Valve 202    on/off* off       on    off                                      ______________________________________                                         *Valve 101 and 202 were closed alternatively.                            

After switching off a pump a period of 10 seconds was used to allowcells to settle, except after pumping cells into the system (period 1),for which 45 seconds settling time was used. However, these periods maybe varied.

RESULTS AND CONCLUSIONS

It is apparent that at the left side of the chamber all cells areviable, whilst on the right side cells all are non-viable. This is insharp distinction with the middle of the chamber where all cells aremixed. As expected, the separation improved when going further away fromthe center of the chamber, and a substantially complete (approximately100%) separation of viable and non-viable cells was achieved at the exitof the chamber. This is in contrast with batch separation that werepreviously performed (Marks et al., 1993) and with which a 90-95%separation was achieved.

It is estimated that nearly complete separation was achieved at adistance of 3 cm from the point of inflow. This implies that as thechamber is 30 cm long it has a minimum estimated total of 5 idealseparation steps. In reality this is probably more as the flow near thepoint of inflow is not well defined, and will be better defined furtheraway from it. For a fluid flow of 0.55 ml/min it took an estimated 2hours to travel from the point of inflow to the outflow.

The use of this system for the separation of other cell types, inparticular plant protoplasts. Friend Murine erythroleukamic cells anddifferent species of bacteria is presently under investigation.

It will be appreciated that variation may be made to the above-mentionedembodiments and methods without departing from the scope of theinvention. For example, it is understood that variation to theconductivity or relative permittivity of a suspending medium (such as asolvent or liquid) may be made so as to alter the effects of the DEPforces on particles experiencing the DEP effects. Similarly variation tothe size and shape of electrode geometry may be made in order to permithigh field gradients to be obtained, thus facilitating local confinementof two (or more) particle types within a generally small region.

Thus by varying the aforementioned characteristics and by carefulselection of frequency applied to establish the DEP field, a high degreeof selection of different species is possible.

Although described in the specific embodiments as having a relativelysmall area, it is envisaged that large electrode arrays may be assembledhaving a total area of 0.1-1 m². Such electrode arrays would permit arelatively large throughput of liquid medium, for example of the orderof litres or tens of litres per minute. Similarly arrays of electrodescould be manufactured such that they lie above one another, therebycreating a three dimensional array.

Although reference has been made to a chamber wherein pressure urgesliquid, supporting a mixture of particles to be separated, through thechamber, the invention may also be used as a dielectrophoretic column toseparate several different species whose dielectrophoretic propertiesare similar. The invention configured to operate in this manner may beenvisaged as performing separation by dielectrophoresis, in a similarmanner as a chemical separator, such as a gas chromatograph. The controlmeans is arranged to operate so as to pulse the supporting mediumthrough the chamber when the field is activated.

Experiments using a 0.25 ml mixture of micrococcus lysodeikticus(Gram+ve), Escherichia coli (Gram -ve) and Saccharomyces cerevisiaesuspended in 280 mM mannitol of conductivity 50 ms/m (adjusted using 1MNaCl) were allowed to pass through (an initially "loaded") columncomprising two sets of castellated, interdigitated, microelectrodesforming one side of chamber of a column 0.25 ml. A 4-8 V (peak to peak)voltage signal was applied at 50 kHz (or 10˜100 kHz). The yeast cellswere collected first (˜0.3 ml fraction) from the column. The E-coli werecollected next (identified by lack of Gram staining in morphology);whilst the M. lysodeikticus were retained and later collected byflushing through chamber with the voltage removed. Thus continuousseparation of three different species was possible.

It will also be appreciated that the invention is particularly effectivewhen used to separate cellular matter, when the cellular matter islabelled. For example fluorescent labels such as Fluoresceinisothiocyanate (FITC), gold or other chemical labels, cause variation inthe conductance and/or permittivity of cellular matter. Careful choiceof labels; electrical properties of the supporting fluid; and thefrequency of applied electric fields, give rise to enhanced separation.

The invention has been described with specific reference to cellularmatter. However, separation of non-cellular matter may also be achievedby using the invention.

Similarly coatings on the electrodes may enhance/inhibit chemicalreactions. The coating(s) may comprise hydrophobic or hydrophilicchemicals, acidic or basic chemicals or antibodies. The fact thatparticles are confined by DEP forces enhances rates of reaction.

We claim:
 1. An apparatus for separating first particles and secondparticles from a fluid supporting said first particles and said secondparticles, said apparatus comprising:a housing forming a chamber, saidhousing having formed thereon an inlet, a first outlet, and a secondoutlet, said inlet and said first and said second outlets communicatingwith sald chamber; a first electrode array and a second electrode arraydisposed within said chamber formed by said housing; a fluid flow systemsupplying said fluid to said Inlet and removing said fluid from saidfirst and said second outlets, thereby operating a fluid flow withinsaid chamber; frequenoy generating means connected to said first andsaid second electrode arrays for establishing a dielectrophoretic fieldbetween said first and said second electrode arrays to cause a firstresultant force to be experienced by said first particles; and controlmeans for controlling said frequency generating means and said fluidflow system to remove said fluid from said first outlet while said firstresultant force is experienced by said first particles, said controlmeans including means to change said dielectrophoretic field to cause asecond resultant force to be experienced by said second particles.
 2. Anapparatus acoording to claim 1, wherein said fluid flow systemincludes:a conduit forming a fluid carrying passage, said conduit beingcoupled to said housing via said inlet; and a selectively operablepressure source coupled to said conduit so as to provide said fluid tosaid inlet via said conduit.
 3. An apparatus according to claim 1,wherein said fluid flow system further comprises:a second conduitcoupled to said housing via said first outlet; a third couduit coupledto said housing via said second outlet; and at least one valve disposedin one of said second conduit and said third conduit.
 4. An apparatusaccording to any of claims 1, 2 or 3, wherein:said fluid flow systemfurther comprises a gravity feed.
 5. An apparatus according to any ofclaims 1, 2 or 3, wherein:said fluid flow system further comnpries apump.
 6. An apparatus according to claim 2, wherein said fluid flowsystem further comprises:a second conduit forming a first fluid carryingpassage coupled to said housing via said first outlet; a third conduitforming a second fluid carrying passage coupled to said housing via saidsecond outlet; at least one valve disposed in one of said second conduitand said third conduit, wherein said control means includes amicroprocessor adapted to activate said dielectrophoretlo field, said atleast one valve, and said pressure source in synchronism.
 7. Anapparatus according to claim 2, wherein:each of said first and saidsecond electrode arrays comprises sub-groups of electrodes, and saidcontrol means controls said frequency generating means to cause saidsub-groups of said first electrode array and said sub-groups of saidsecond electrode array to be actuated in a cyclic manner.
 8. Anapparatus according to claim 1, further comprising:means for varying apotential difference between adjacent electrodes of said first electrodearmy and said second electrode array.
 9. An apparatus according to claim1, further comprising:means for varying a frequency of voltage appliedbetween adjacent electrodes of said first electrode array and saidsecond electrode array.
 10. An apparatus according to claim 1,wherein:said first electrode array and said second electrode array areinterdigitated; castellated electrodes.
 11. An apparatus according toclaim 1, wherein:one of said first particles and said second particlesis live cellular matter, and the other of said first particles and saidsecond particles is dead cellular matter, and said apparatus is arrangedto separate said live cellular matter from said dead cellular matter.12. An apparatus according to claim 1, wherein:at least one of saidfirst electrode array and said second electrode array includes: anelectrical contact for connection to an electrical energy source havinga polarity which is controlled to change, and a surface adapted for usein said chamber.
 13. An apparatus according to claim 1, wherein:at leastone of said first electrode aray and said second electrode array iscoated with a substance to perform at least one of enhance and inhibit achemical reaction.
 14. An apparatus for separating first particles andsecond particles from a fluid supporting said first particles and saidsecond particles, said apparatus comprising:a housing forming a chambercapable of holding said fluid, said housing forming an inlet forallowing said fluid to flow into said chamber, a first outlet forallowing one of said first particles and said second particles to flowout of said chamber, and a second outlet for allowing a remaining one ofsaid first particles and said second particles to flow out of saidchamber; a first electrode array disposed in said chamber and beingcapable of contacting said fiuld; a second electrode array disposed insaid chamber and being capable of contacting said fluld; a fluid flowsystem supplying said fluid to said inlet and removing said fluid fromsaid first and said second outlets, thereby creating a fluid flow withinsaid chamber; a frequency source operatively coupled to said first andsaid second electrode arrays and adapted to establish adielectrophoretic field between said first electrode army and saidsecond electrode array; control means for controlling said frequencysource, said control means providing that said dielectrophoretic fieldincludes a first dielectrophoretic force and a second dielectrophoreticforce in an alternating fashion, said first dielectrophoretic forcebeing experienced by at least one of sald first particles and saidsecond particles, and said second dielectrophoretic force beingexperienced by at least one of said first particles and said secondparticles.
 15. An apparatus according to claim 14, wherein:said firstelectrode array and said second electrode array are interdigitated,castellated electrodes.
 16. An apparatus according to claim 14, whereinsaid frequency source is a single frequency generator.
 17. A method forselectively separating first type particles from second type particles,said method comprising:flowing a fluid containing said first typeparticles and said second type particles into a chamber formed by ahousing, said chamber having a first and a second electrode arraydisposed therein, said housing forming a first inlet, a second inlet, athird inlet, a first outlet, and a second outlet, said fluid flowinginto said chamber through said first inlet; activating a power source toestablish a dielectrophoretic field between said first electrode arrayand said second electrode array to cause a first resultant force to beexperienced by said first type particles and flowing a second fluid,containing none of said first type and said second type particles,through said second inlet to cause said second type particles to move ina first directlon, said second fluid exiting said chamber through saidfirst outlet; deactivating said power source and flowing said secondfluid through said third inlet to cause said first type particles andsaid second type particles to move in said second direction, said seconddirection being in a direction opposite to said first direction, saidsecond fluid exiting said chamber through said second outlet; andreapplying said power source to reestablish said dielectrophoretic fieldbetween said first electrode array and said second electrode array; andflowing said second fluid through said second inlet to cause said secondtype particles to move in said first direction, said second fluidexiting said chamber through said first outlet.
 18. A method accordingto claim 17, wherein:said fluid flows into said chamber through saidfirst inlet by an action of a first pump, said fluid flows into saidchamber through said second inlet by an action of a second pump, andsaid fluid flows into said chamber through said third inlet by an actionof a third pump.
 19. A method acoording to claim 17 or 18, wherein eachof said first and said second electrode arrays comprises sub-groups ofelectrodes, and said dielectrophoretic field is changed by activatingsaid sub-groups of said first electrode array and said sub-groups ofsaid second electrode array in a cyclic manner.
 20. A method accordingto claim 18, wherein when said fluid flows through said chamber, saidflowing is accomplished by urging said fluid through said chamber usinga pressure source.
 21. A method according to claim 20, wherein:at leastone of said second type particles and said first-type particles areremoved from said chamber using said pressure source.
 22. A methodaccording to claim 17, further comprising:labelling at least one of saidfirst type particles and said second type particles of one of on andbefore entering said chamber in order to enhance separation of saidsecond type particles from said first type particles.
 23. A methodaccording to claim 22, wherein;said step of labeling uses gold to labelsaid at least one of said first type particles and said second typeparticles.